Systems metabolic engineering of Corynebacterium glutamicum for efficient production of L-isoleucine
L-Isoleucine is an essential branched-chain amino acid for livestock and poultry, supporting protein accretion and regulating energy metabolism, immune function, and stress resilience. A genetically stable L-isoleucine producer, Corynebacterium glutamicum cgl-Ile0, was obtained via biosensor-assisted ARTP mutagenesis, and produced 11.52 g/L L-isoleucine in a 5-L fermenter, with a yield of 0.11 g/g and a productivity of 0.24 g/L/h. Whole-genome resequencing revealed four mutations associated with the phenotype, including aspBV346I, asdP27E, brnEL87S, and brnFR28P. Structure-guided protein engineering of two rate-limiting enzymes—threonine dehydratase (TD) and acetohydroxyacid synthase (AHAS)—generated strain cgl-Ile1, increasing titer, yield, and productivity by 39.50%, 27.27%, and 37.50%, respectively, relative to strain cgl-Ile0. Subsequent modular optimization of L-isoleucine biosynthesis, oxaloacetate supply module, cofactor-supply module, and transport/export module yielded the final strain cgl-Ile8. Through optimization pH and dissolved oxygen, the titer, yield and productivity of L-isoleucine produced by strain cgl-Ile8 in a 5-L fermenter were 48.49 g/L, 0.31 g/g and 1.01 g/L/h, which were 4.21-, 2.82-, and 4.21-fold those of strain cgl-Ile0, respectively. Scale-up to a 50-L fermenter further increased the titer, yield and productivity to 50.12 g/L, 0.32 g/g and 1.04 g/L/h, respectively, representing the highest reported L-isoleucine titer in C. glutamicum to date. We developed an industrial L-isoleucine-producing C. glutamicum strain by integrating biosensor-guided ARTP mutagenesis, structure-guided protein engineering, and modular pathway rewiring, providing a practical and transferable framework for constructing GRAS amino-acid producers for animal nutrition.